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Definitions

• the present invention relates to novel polypeptides which are variants of Bile Salt-Stimulated Lipase (BSSL; EC 3.1.1.1). It also relates to DNA molecules encoding the said polypeptides, and to subproducts comprising the said DNA molecules.
• the invention further relates to processes for producing the said BSSL variants and for producing transgenic non-human mammals capable of expressing the BSSL variants. Furthermore the invention relates to such transgenic animals as well as to infant formulas comprising milk from such transgenic animals.
• the invention also relates to pharmaceutical compositions comprising the said polypeptides; and the use of the said polypeptides and DNA molecules for the manufacture of medicaments.
• triacylglycerols constitute more than 95% of these lipids.
• Some of the lipids e.g. certain fatty acids and the fat-soluble vitamins, are essential dietary constituents.
• the triacylglycerols as well as the minor components, i.e. esterified fat-soluble vitamins and cholesterol, and diacylphosphatidylglycerols, require hydrolysis of the ester bonds to give rise to less hydrophobic, absorbable products. These reactions are catalyzed by a specific group of enzymes called Upases.
• the essential lipases involved are considered to be Gastric Lipase, Pancreatic Colipase-Dependent Lipase (hydrolysis of tri- and diacylglycerols), Pancreatic Phospholipase A2 (hydrolysis of
• Bile Salt-Stimulated Lipase plays an essential part in the hydrolysis of several of the above mentioned lipids. Together with bile salts the products of lipid digestion form mixed micelles or unilamellar vesicles (Hernell et al., 1990) from which absorption occurs. Bile Salt-Stimulated Lipase
• Bile Salt-Stimulated Lipase is a constituent of milk in a limited number of species, e.g. humans, gorillas, cats and dogs (Hernell et al., 1989, Hamosh et al., 1986). When mixed with bile in upper small intestinal contents, BSSL is specifically activated by primary bile salts (Hernell, 1975). BSSL, which accounts for approximately 1% of total milk protein
• BSSL is a non-specific lipase (EC 3.1.1.1) in as much as it hydrolyses not only triacylglycerol but also di- and monoacylglycerol, cholesteryl esters and fat-soluble vitamin esters (Blackberg & Hernell, 1983).
• BSSL has the potential to hydrolyze most human milk lipids by itself, albeit the most efficient utilization of human milk triacylglycerol requires the synergistic action of gastric lipase (EC 3.1.1.3), colipase- dependent pancreatic lipase (EC 3.1.1.3), and BSSL (Bernback et al., 1990).
• BSSL is a single-chain glycoprotein.
• the deduced protein (SEQ ID NO:3) contains 722 amino acid residues and is highly glycosylated (Abouakil et al., 1989).
• the N-terminal half of the protein shows a striking homology to acetyl cholinesterase and some other esterases (Nilsson et al., 1990).
• a tentative active site serine residue is located at serine-194; the sequence around this serine accords with the consensus active-site sequence of serine hydrolases.
• the single tentative N-glycosylation site is positioned only seven residues N-terminal of the active site serine (Nilsson et al., 1990).
• the BSSL sequence contains in its C-terminal part 16 proline-rich repeats of 11 amino acid residues each. A variation in number of repeats seems to be a major explanation for differences in molecular size and amino acid composition between corresponding enzymes from different species (Han et al., 1987, Fontaine et al., 1991, Kyger et al., 1989). These repeats carry most of the 15-20% carbohydrate of the protein (Baba et al., 1991, Abouakil et al., 1989).
• lipid malabsorption and hence malnutrition, are reduced intraluminal levels of Pancreatic Colipase-Dependent Lipase and/or bile salts.
• Typical examples of such lipase deficiency are patients suffering from cystic fibrosis, a common genetic disorder resulting in a lifelong deficiency in 80% of the patients, and chronic pancreatitis, often due to chronic alcoholism.
• pancreatic lipase The present treatment of patients suffering from a deficiency of pancreatic lipase is the oral administration of very large doses of a crude preparation of porcine pancreatic enzymes.
• Colipase-Dependent Pancreatic Lipase is inactivated by the low pH prevalent in the stomach. This effect cannot be completely overcome by the use of large doses of enzyme.
• the large doses administered are inadequate for most patients, and moreover the preparations are impure and unpalatable.
• Certain tablets have been formulated which pass through the acid regions of the stomach and discharge the enzyme only in the relatively alkaline environment of the jejunum. However, many patients suffering from pancreatic disorders have an abnormally acid jejunum and in those cases the tablets may fail to discharge the enzyme.
• Recombinant BSSL variants according to the invention have maintained catalytic activity, but contain less glycosylation sites than full-length BSSL, and are thus produced with a potentially reduced degree of carbohydrate heterogeneity.
• This reduced complexity facilitates purification and characterization of the recombinant protein, which will result in a more cost-effective production of polypeptides having BSSL activity.
• the reduced degree of glycosylation is less demanding for the host and allows higher production in several host cells.
• the reduced number of glycosylation sites in a BSSL variant allows efficient production in lower eukaryotes and restricts the potential risk of abberrant glycosylation, which may raise immunological reactions.
• the reduced size and less complex glycosylation also implies that the host range is broader than for a protein having very complex and heavy carbohydrate moieties.
• a further possible advantage with a recombinant BSSL variant lacking most or all of the O-glycosylated repeats is a reduced risk for an immunological response in the recipient individual. This is due to the fact that the O-linked sugar may be very heterogenous depending on the cell in which it is produced.
• a BSSL variant which is selected for having a reduced uptake will be active on the dietary lipid substrates for a longer period of time, leading to a more efficient intraluminal digestion. Examples of such variants are molecules with reduced
• BSSL has been suggested to be of particular importance for the utilization of long-chain polyunsaturated fatty acids (Hernell et al., 1993), which are of great importance for neuro-development of the newborn infant, and of vitamin A.
• a BSSL variant according to the invention which is more effective in these respects, can be selected by known methods.
• a tnmcated, or shortened, enzyme is likely to be different with regard to conformation which may affect the specificity against different lipid substrates.
• the invention relates to a nucleic acid molecule encoding a polypeptide which is a BSSL variant shorter than 722 amino acids, said BSSL variant comprising part of the amino acid sequence shown as residues 536-722 in SEQ ID NO: 3.
• part of the amino acid sequence is to be understood as comprising one single amino acid as well as a sequence of several amino acids or several such sequences combined.
• BSSL variant is to be understood as a polypeptide having BSSL activity and comprising a part of the amino acid sequence of human BSSL shown as SEQ ID NO: 3 in the Sequence Listing.
• polypeptide having BSSL activity is to be understood as a polypeptide comprising at least the properties (a) suitable for oral administration;
• pancreatic proteases e.g. trypsin
• bile salts are present
• the invention relates to a nucleic acid molecule according to above, wherein the said BSSL variant has a phenylalanine residue in its C-terminal position, or comprises the sequence Gln-Met-Pro in its C-terminal part, alternatively comprises the amino acid sequence shown as residues 712-722 in SEQ ID NO: 3 in its C-terminal part.
• C-terminal position designates the position of the final C-terminal residue
• C-terminal part is to be understood as the approximately 50 amino acid residues which constitute the C-terminal end of the BSSL variant.
• the invention further relates to a nucleic acid molecule according to above, wherein the said BSSL variant comprises less than 16 repeat units.
• the term "repeat unit” designates one of the repeated units of 33 nucleotides each which are indicated in SEQ ID NO: 1 in the
• the invention relates to a nucleic acid molecule according to above which encodes a polypeptide, the amino acid sequence of which is at least 90% homologous with the amino acid sequence shown as SEQ ID NO: 5, 6 or 9 in the Sequence Listing, as well as a nucleic acid molecule which encodes a polypeptide, the amino acid sequence of which is at least 90% homologous with the amino acid sequence shown as SEQ ID NO: 7 in the Sequence Listing, with the exception for those nucleic acid molecules which encode polypeptides which have an asparagine residue at position 187.
• the invention also relates to a polypeptide shown as SEQ ID NO: 5, 6, 7 or 9 in the Sequence Listing, as well as a polypeptide encoded by a nucleic acid sequence according to above.
• the invention further relates to a hybrid gene comprising a nucleic acid molecule according to above, a replicable expression vector comprising such a hybrid gene, and a cell harbouring such a hybrid gene.
• This cell may be a prokaryotic cell, a unicellular eukaryotic organism or a cell derived from a multicellular organism, e.g. a mammal.
• hybrid gene denotes a nucleic acid sequence comprising on the one hand a nucleic acid sequence encoding a BSSL variant as defined above and on the other hand a nucleic acid sequence of the gene which is capable of mediating the expression of the hybrid gene product.
• gene denotes an entire gene as well as a subsequence thereof capable of mediating and targeting the expression of the hybrid gene to the tissue of interest. Normally, said subsequence is one which at least harbours one or more of a promoter region, a transcriptional start site, 3' and 5' non-coding regions and structural sequences.
• the hybrid gene is preferably formed by inserting in vitro the nucleic acid sequence encoding the BSSL variant into the gene capable of mediating expression by use of techniques known in the art.
• the nucleic acid sequence encoding the BSSL variant can be inserted in vivo by homologous recombinantion.
• the term "replicable" means that the vector is able to replicate in a given type of host cell into which it has been introduced.
• Immediately upstream of the nucleic acid sequence there may be provided a sequence coding for a signal peptide, the presence of which ensures secretion of the BSSL variant expressed by host cells harbouring the vector.
• the signal sequence may be the one naturally associated with the nucleic acid sequence or of another origin.
• the vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
• the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromosome or virus.
• the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome(s) into which it has been integrated. Examples of suitable vectors are a bacterial expression vector and a yeast expression vector.
• the vector of the invention may carry any of the nucleic acid sequences of the invention as defined above.
• the invention in another aspect, relates to a process for the production of a recombinant polypeptide, said process comprising (i) inserting a nucleic acid molecule according to above in a hybrid gene which is able to replicate in a specific host cell or organism; (ii) introducing the resulting recombinant hybrid gene into a host cell or organism; (iii) growing the resulting cell in or on a culture medium, or identifying and reproducing an organism, for expression of the polypeptide; and (iv) recovering the polypeptide.
• the medium used to grow the cells may be any conventional medium suitable for the purpose.
• a suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above.
• the methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA.
• the recombinant human BSSL variant expressed by the cells may be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.
• the BSSL variant is produced intracellularly by the recombinant host, that is, is not secreted by the cell, it may be recovered by standard procedures comprising cell disrupture by mechanical means, e.g. sonication or homoger ⁇ zation, or by enzymatic or chemical means followed by purification.
• the DNA sequence encoding the BSSL variant should be preceded by a sequence coding for a signal peptide, the presence of which ensures secretion of the BSSL variant from the cells so that at least a significant proportion of the BSSL variant expressed is secreted into the culture medium and recovered.
• the invention also relates to an expression system, comprising a hybrid gene which is expressible in a host cell or organism harbouring said hybrid gene, so that a recombinant polypeptide is produced when the hybrid gene is expressed, said hybrid gene being produced by inserting a nucleic acid sequence according above into a gene capable of mediating expression of the said hybrid gene.
• a possible process for producing a recombinant BSSL variant of the invention is by use of transgenic non-human mammals capable of excreting the BSSL variant into their milk.
• transgenic non-human mammals has the advantage that large yields of the recombinant BSSL variant are obtainable at reasonable costs and, especially when the non- human mammal is a cow, that the recombinant BSSL variant is produced in milk which is the normal constituent of, e.g., infant formulae so that no extensive purification is needed when the recombinant BSSL variant is to be used as a nutrient supplement in milk-based products.
• production in a higher organism such as a non-human mammal normally leads to the correct processing of the mammalian protein, e.g. with respect to post-translational processing as discussed above and proper folding. Also large quantities of a substantially pure BSSL variant may be obtained.
• the expression system referred to above may be a mammalian expression system comprising a DNA sequence encoding a BSSL variant inserted into a gene encoding a milk protein of a non-human mammal, so as to form a hybrid gene which is expressible in the mammary gland of an adult female of a mammal harbouring said hybrid gene.
• the mammary gland as a tissue of expression and genes encoding milk proteins are generally considered to be particularly suitable for use in the production of heterologous proteins in transgenic non-human mammals, as milk proteins are naturally produced at high expression levels in the mammary gland. Also, milk is readily collected and available in large quantities.
• the use of milk protein genes in the production of a recombinant BSSL variant has the further advantage that it is produced under conditions similar to the its natural production conditions in terms of regulation of expression and production location (the mammary gland).
• the hybrid gene referred to above preferably comprises a sequence encoding a signal peptide so as to enable the hybrid gene product to be secreted correctly into the mammary gland.
• the signal peptide will typically be the one normally found in the milk protein gene in question or one associated with the DNA sequence encoding the BSSL variant. However, also other signal sequences capable of mediating the secretion of the hybrid gene product to the mammary gland are relevant. Of course, the various elements of the hybrid gene should be fused in such a manner as to allow for correct expression and processing of the gene product. Thus, normally the DNA sequence encoding the signal peptide of choice should be precisely fused to the N- terminal part of the DNA sequence encoding the BSSL variant. In the hybrid gene, the DNA sequence encoding the BSSL variant will normally comprise its stop codon, but not its own message clenature and
• the polyadenylation site Downstream of the DNA sequence encoding the BSSL variant, the mRNA processing sequences of the milk protein gene will normally be retained. A number of factors are contemplated to be responsible for the actual expression level of a particular hybrid gene. The capability of the promoter as well of other regulatory sequences as mentioned above, the integration site of the expression system in the genome of the mammal, the integration site of the DNA sequence encoding the BSSL variant in the milk protein encoding gene, elements conferring post-transcriptional regulation and other similar factors may be of vital importance for the expression level obtained. On the basis of the knowledge of the various factors influencing the expression level of the hybrid gene, the person skilled in the art would know how to design an expression system useful for the present purpose.
• the milk protein gene to be used may be derived from the same species as the one in which the expression system is to be inserted, or it may be derived from another species.
• the regulatory elements that target gene expression to the mammary gland are functional across species boundaries, which may be due to a possible common ancestor (Hennighausen et al., 1990).
• subsequences thereof to be used in the construction of an expression system of the invention are normally found among whey proteins of various mammalian origins, e.g. a whey acidic protein (WAP) gene, preferably of murine origin, and a ⁇ -lactoglobulin gene, preferably of ovine origin.
• WAP whey acidic protein
• casein genes of various origins may be found to be suitable for the transgenic production of a BSSL variant, e.g. bovine ⁇ Sl-casein and rabbit ⁇ -casein.
• the presently preferred gene is a murine WAP gene as this has been found to be capable of providing a high level of expression of a number of foreign human proteins in milk of different transgenic animals (Hennighausen et al, 1990).
• Another sequence preferably associated with the expression system of the invention is a so-called expression stabilizing sequence capable of mediating high-level expression. Strong indications exist that such stabilizing sequences are found in the vicinity of and upstreams of milk protein genes.
• Included in the invention is also a process of producing a transgenic non- human mammal capable of expressing a BSSL variant, comprising (a) introducing an expression system according to above into a fertilized egg or a cell of an embryo of a non-human mammal so as to incorporate the expression system into the germline of the mammal and (b) developing the resulting introduced fertilized egg or embryo into an adult female non- human mammal.
• the incorporation of the expression system into the germline of the mammal may be performed using any suitable technique, e.g. as described in "Manipulating the Mouse Embryo"; A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1986.
• a few hundred molecules of the expression system may be directly injected into a fertilized egg, e.g. a fertilized one cell egg or a pro-nucleus thereof, or an embryo of the mammal of choice, and the microinjected eggs may then be transferred into the oviducts of pseudopregnant foster mothers and allowed to develop.
• the process of producing a transgenic non-human mammal capable of expressing a BSSL variant can also comprise a process wherein the said mammal is substantially incapable of expressing BSSL from the mammal itself.
• Such a process comprises (a) destroying the BSSL expressing capability of the mammal so that substantially no mammalian BSSL is expressed and inserting an expression system according to above into the germline of the mammal in such a manner that a BSSL variant is expressed in the mammal; and /or (b) replacing the mammalian BSSL gene or part thereof with an expression system as defined above.
• the mammalian BSSL expressing capability can conveniently be destroyed by introduction of mutations in the DNA sequence responsible for the expression of BSSL.
• Such mutations may comprise mutations which make the DNA sequence out of frame, introduction of a stop codon, or a deletion of one or more nucleotides of the DNA sequence.
• the mammalian BSSL gene or a part thereof may be replaced with an expression system as defined above or with a DNA sequence encoding the BSSL variant by use of the well known principles of homologous recombination.
• the invention relates to a transgenic non- human mammal harbouring in its genome a DNA sequence according to above.
• the said DNA sequence can preferably be present in the germline of the mammal, and in a milk protein gene of the mammal.
• the transgenic non-human mammal can preferably be selected from the group consisting of mice, rats, rabbits, sheep, pigs and cattle.
• the invention further relates to an infant formula comprising milk according to above, and an infant formula comprising a BSSL variant as defined above.
• the infant formula may be prepared using conventional procedures and contain any necessary additives such as minerals, vitamins etc.
• the invention relates to a pharmaceutical composition comprising a BSSL variant as defined above, as well as such a BSSL variant for use in therapy.
• the invention relates to the use of a BSSL variant as defined above for the manufacture of a medicament for the treatment of a pathological condition related to exocrine pancreatic insufficiency; cystic fibrosis; chronic pancreatitis; fat malabsorption; malabsorption of fat soluble vitamins; fat malabsorption due to physiological reasons.
• the invention also relates to the use of a BSSL variant for the manufacture of a medicament for the improvement of the utilization of dietary lipids, particularly in preterm born infants.
• plasmid pS146 containing the 2.3 kb human BSSL cDNA (Nilsson et al., 1990) cloned into pUC19 was digested with Hindlll and Sail and the BSSL cDNA was introduced into a bovine papilloma virus (BPV) expression vector, pS147 (Fig. 1).
• This vector contains the human BSSL cDNA under control of the murine metallothioneine 1 (mMT-1) enhancer and promoter element (Pavlakis & Hamer, 1983).
• the mRNA processing signals are provided by a genomic fragment containing part of exon II, intron II, exon III and downstream elements of the rabbit ⁇ -globin gene. This transcriptional unit was cloned into a vector containing the entire BPV genome. Transcription was unidirectional for BPV and the BSSL
• the vector For propagation of the vector in E.coli the vector also contains pML2d, a pBR322 derivative (Sarver et al., 1982).
• the expression vector pS147 was co-transfected with a vector encoding the neomycin resistance gene driven by the Harvey Sarcoma virus 5'-Long terminal repeat and Simian virus 40 polyadenylation signals (Lusky & Botchan, 1984).
• the BSSL cDNA was subdoned as a Ndel- BamHI fragment from plasmid pT7-7 (Ausubel et al., 1992) into plasmid pGEMEX-1 (Promega, Madison, WI, USA) (Studier & Moffat, 1986). By this doning procedure the T7 gene 10 encoding sequence was replaced by the BSSL gene coding for the mature protein preceded by a start codon.
• the final expression vector, pGEMEX/BSSL was verified by DNA sequencing using spedfic BSSL internal primers.
• Nudeotide number 1 was assigned to the A in the initiation codon ATG. For amino add numbering the first methionine in the signal peptide is -23 and the first amino add residue of the mature protein, an alanine, is assigned number 1.
• PCR-1 and PCR-2 were synthesized, PCR-1 and PCR-2 (Table 1).
• the Hmdfll, Sail and BamHI sites were created for cloning into different plasmids.
• the Bell site was generated in the BSSL sequence without altering the amino add sequence. This was done to facilitate addition of synthetic DNA to obtain the other variants.
• the primer PCR-2 contains two synthetic stop codons.
• the resulting PCR fragments were digested with BamHI and Hfndlll and doned into pUC18 for sequence analysis. This plasmid was designated pS157.
• the correct PCR fragment was inserted into the BPV expression vector by fusion to the BSSL sequence at the unique Asp700 site (position 1405 in the BSSL cDNA) and the SalI site in front of the ⁇ -globin gene fragment, resulting in pS257.
• the B-variant construction (SEQ ID NO: 5) was done using
• oligonudeotides encodes the very C-terminal amino add sequence, representing lysine 712 to phenylalanine 722 in the full-length protein. This fragment was fused to glutamine 535. A translational stop was inserted directly after the last phenylalanine. This fragment contains a Bell site in the 5'-end and a Sail site in the 3'-end, allowing introduction into pS157. The resulting plasmid was digested with Asp700 and Sail and the 313 bp fragment was introduced into the expression vector as described above. The resulting plasmid was designated pS258.
• Synthetic oligonudeotides used for construction of the BSSL variants Nudeotides of restriction sites are underlined. Translational stop signals are indicated by bold letters. The altered codon in variant N is indicated in PCR-3 by bold letters and an asterisk.
• oligonudeotides 1 to 6 (Table 1) were used.
• the annealed DNA fragment contains two repetitions, encoding eleven amino adds, identical to consensus (Nilsson et al., 1990), inserted between glutamine 535 and the lysine 712 to phenylalanine 722 sequence.
• This fragment also contains a Bell site in the 5' -end and a Sail site in the 3'-end, allowing the same doning strategy as above.
• the resulting plasmid was designated pS259.
• variant N non-N-glycosylated variant, SEQ ID NO: 7
• two PCR primers PCR-3 and PCR-4 in Table 1
• the EcoRI and BamHI sites were created for doning of the 360 bp PCR product into pUC19 for sequence analysis.
• the potential N-linked glycosylation site at asparagine 187, was changed to a glutamine.
• the modified sequence was isolated as a BaE-Hin dlll fragment and doned into Sacl and HmdIII digested pUC19 together with a Sacl and Ball fragment containing the mMT-1 promoter and 5'-end of BSSL cDNA.
• An approximately 1.2 kb Sacl- Dra ⁇ ll fragment was isolated from this plasmid and inserted in the mMT-1 element and BSSL cDNA sequence, respectively, within the expression vector.
• the resulting plasmid was designated pS299.
• the vectors were co-transfected into the murine cell line C127 (ATCC CRL 1616) according to the caldum-phosphate predpitation method (Graham & Van der Eb, 1973).
• Neomydn resistant cell dones were selected with 1.5 mg x ml -1 of G418 and after 10- 15 days resistant cell dones were isolated from the master plates and passaged for analysis. 1.1.4. Bacterial strains and culture conditions
• the vector pGEMEX/BSSL was transformed into E.coli strains JM109(DE3) and BL21(DE3)pLysS. The expression experiments were carried out as described by Studier et al. (1986). After harvesting of bacteria, the cells were pelleted by centrifugation (5,000 x g for 10 min at 4°C). For preparation of periplasm- and cytoplasm fractions, the pellet was resuspended in 4 ml 20 mM Tris-Cl/20% sucrose, pH 8.0, 200 ul 0.1 M EDTA and 40 ul lysozyme (15 mg/ml in water) per gram of pellet. The suspension was incubated on ice for 40 minutes.
• the cells were suspended in 40 mM Tris-Cl, 0.1 mM EDTA, 0.5 mM phenylmethylsulphonylfluoride, pH 8.2, freeze-thawed and sonicated several times to lyse. The cell lysate was centrifuged (30,000 x g for 30 min at 25°C).
• RNA and DNA were prepared from isolated mammalian cell lines or E.coli cells (Ausubel et al., 1992). The RNA or DNA were fractionated on agarose gels and blotted onto GeneScreen Plus (New England Nudear) and hybridized according to the supplier's instructions.
• Bile salt-stimulated lipase was purified from human milk as previously described (Blackberg & Hernell, 1981). The purified preparation was homogenous as judged by SDS-PAGE and had a specific activity of 100 ⁇ mol fatty add released x min -1 and mg -1 when assayed with long-chain triacylglycerol as substrate. 1.1.7. Enzyme assay
• the enzyme assay was as described (Blackberg & Hernell, 1981) using triolein emulsified with gum arabic as substrate. The incubations were carried out with 10 mM sodium cholate as activating bile salt. When the bile salt dependency was tested bile salts (sodium cholate or sodium deoxycholate, Sigma Chem. Co.) were added to the concentrations given in Fig. 3. 1.1.8. Western blotting
• Sepharose (Pharma ⁇ a LKB Biotechnology). The respective media were mixed with Blue Sepharose (approx 10 ml of medium per ml of gel). The gel was washed with (10 ml per ml of gel) with 0.5 M Tris-Cl buffer, pH 7.4, containing 0.1 M KCl. The enzyme activity was eluted with 1.5 M KCl in the same buffer. By this procedure a 25-30-fold concentration was obtained as well as a 3-5-fold purification. SDS-PAGE was performed on 10% polyacrylamide gels essentially according to Laemmli (1970). After transfer to nitrocellulose membranes and incubation with a polydonal rabbit antiserum to purified BSSL detection was made using goat anti- rabbit IgG conjugated with alkaline phosphatase and a developing kit from Bio-Rad.
• variant B containing a BSSL activity of 2.5 ⁇ mol fatty add released x min -1 , 1 ⁇ l of 1 M ⁇ -mercaptoethanol and 0.5 ⁇ l of 10% (w/v) SDS was added. After boiling for 5 min, 10 ⁇ l 0.1 M Na-phosphate buffer, pH 8.0, 6 ul 0.1 M EDTA, 4 ul 7.5% (w/v) Nonidet P 40 and 5 ul (1U) N- glycosidase F (Boehringer Mannheim) were added. As a control the same amount of variant B was treated identically except that no glycosidase was added. After an overnight incubation at 37°C, the samples were run on SDS-PAGE and blotted using the polyclonal rabbit BSSL antiserum. 1.2. RESULTS
• variant C SEQ ID NO: 6
• a fragment containing two repeats having a sequence identical to consensus were inserted between glutamine-535 and the lysine-712 to phenylalanine-722 sequence.
• Variant N was obtained by altering the potential N-glycosylation site at asparagine-187 to a glutamine.
• DNA samples were prepared from the cell lines transfected with the expression vectors encoding the different BSSL variants.
• the prepared DNA was digested with BamHI, fractionated on agarose gels and transferred to membranes for hybridization.
• the probe used was 32 P- labelled BSSL cDNA.
• the hybridization results confirmed the presence of the recombinant genes and also that the vector copy number was approximately equal in the different cell lines (Fig. 2).
• the positions of the hybridizing fragments reflected the different lengths of the various BSSL sequences and were in agreement with the expected sizes. The positions were also similar to the bacteria derived DNA used in the transfection experiment, indicating that no major rearrangement of vector DNA had occurred in the cell lines (Fig. 2).
• the upper hybridization signals in the DNA sample representing variant A were probably due to partial digestion. 1.2.3. Expression of mRNA for full-length and mutated BSSL in
• the different lengths of the hybridizing rnRNAs were in accordance with the modifications of the cDNAs.
• the steady state levels of recombinant BSSL mRNA variants in the different samples were about the same except for variant A (Fig. 3).
• the reason for the reduced accumulation of variant A mRNA is not known, but it was observed with two populations of cell lines as well as with isolated clones.
• the presence of equal amounts of RNA in the different samples was confirmed by hybridization to a murine ⁇ -actin probe (Fig. 3, lower panel). 1.2.4. Production of full-length and variants of BSSL in mammalian cells
• Fig. 5A Western blots from clones of the different transfection experiments are shown in Fig. 5A.
• the apparent M r of the BSSL variants were as expected. It should be noted, however, that for full-length BSSL as well as for variants B and C a double band was obtained. Because all three have the single N-glycosylation site intact whereas variant N, which showed no double band, lacks that site, a likely explanation was that the double band resulted from differences in N-glycosylation. Therefore variant B was subjected to digestion with N-glycosidase F. As shown in Fig. 5B, only trace amounts of the upper band remained while the lower band increased in strength indicating that only part of the expressed variant was N- glycosylated.
• BSSL One of the characteristics of BSSL is its specific activation by primary bile salts, e.g. cholate (Hernell, 1975). All the different recombinant forms of BSSL showed the same concentration dependency for cholate activation (Fig. 6). A maximal activity was obtained at about 10 mM in the assay system used. When cholate was exchanged for deoxycholate (a secondary bile salt) no such activation occurred. Thus, the recombinant full-length as well as the different variants showed the same spedfidty regarding bile salt activation. 1.2.5. Expression and biochemical characterization of full-length BSSL in E.coli
• Variant B (SEQ ID NO: 5) lacks all 16 unique, O-glycosylated, proline-rich, C-terminal repeats (aa 536-711) but with the most C-terminal fragment (aa 712-722) fused to glutamine-535.
• Variant C (SEQ ID NO: 6) contains the same C-terminal fragment and two repeats of 11 residues between glutamine-535 and lysine-712.
• variant N non-N-glycosylated variant, SEQ ID NO: 7 the asparagine-187 responsible for the only N- linked sugar was exchanged for a glutamine residue.
• Native BSSL was purified from human milk as described (Blackberg & Hernell, 1981).
• the column was eluted with a gradient 0.05 to 1.0 M NaCl in 5 mM sodium veronal buffer, pH 7.4. Fractions containing lipase activity were pooled and applied to an immunosorbent column. After rinsing with 0.05 M Tris-Cl, pH 7.5, 0.15 M NaCl lipase bound was eluted with 0.1 M glydn-HCl, pH 2.5. The pH of the fractions was immediately adjusted to approx 8 with solid Tris.
• SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
• phenylthiohydantoin 120A analyzer with regular cyde programs and chemicals from the manufacturer. Calculated from a sequenced standard protein ( ⁇ -lactoglobulin) initial and repetitive yields were 47% and 97%, respectively.
• deoxycholate had two effects (Fig. 9B and C). Firstly, it lowered the concentration of cholate needed for activation, and secondly it inhibited enzyme activity at higher bile salt concentration.
• Recombinant BSSL as well as the BSSL variants showed the same pH- stability as native milk BSSL (Fig. 10). An inactivation occured in all cases at a pH around 2.5-3. Above pH 3 all variants were completely stable provided the protein concentration was high enough. This was
• Fig. 11 shows the heat stability of the recombinant enzymes compared to the native milk enzyme.
• variants (B, C, N) appears to be somewhat less stable than the full-length recombinant enzyme and the milk enzyme. However, if the protein concentration was raised by adding bovine serum albumin all variants was stable also at 40°C (Fig. 11).
• the plasmid pS309 contains a Sphl fragment covering the BSSL gene from the 5' untranscribed region to part of the fourth intron.
• the plasmid pS310 contains a Sacl fragment covering a BSSL variant gene sequence from part of the first intron to a part of the sixth intron.
• the deleted sequences are 231 bp which results in a sequence encoding a BSSL variant which has exactly 77 amino adds or seven repeats less than the full-length BSSL.
• the nudeotide sequence of the resulting BSSL variant (“Variant T") is shown in the
• Plasmid pS283 was digested with Ncol and Sacl and a fragment of about 2.7 kb was isolated by electrophoresis.
• Plasmid pS309 was digested with Ncol and BspEl and a fragment of about 2.3 kb containing the 5'-part of the BSSL gene was isolated.
• Plasmid pS310 was digested with BspEl and Sacl and a fragment of about 2.7 kb containing a part of the middle region of the BSSL gene was isolated. These three fragments were ligated and transformed into competent E. coli, strain TG2, and transformants were isolated by ampicillin selection.
• Plasmids were prepared from a number of transformants, and one plasmid, called pS312 (Fig. 14), containing the desired construct was used for further experiments.
• Plasmid pS311 was linearized by partial BamHI digestion. The linearized fragment was isolated and a synthetic DNA linker that converts BamHI to a Sail site (5'- GATCGTCGAC-3'), thereby destroying the BamHI site, was inserted. Since there were two potential positions for integration of the synthetic linker the resulting plasmids were analyzed by restriction enzyme cleavage. A plasmid with the linker inserted at the desired position downstream of exon 11 was isolated and designated pS313.
• Plasmid pS314 contains a genomic fragment from the murine whey acidic protein (WAP) gene (Campbell et al., 1984) doned as a Notl fragment. The genomic fragment has
• URS upstream regulatory sequences
• a unique Kpnl site is located in the first exon 24 bp upstream of the natural WAP translation initiation codon.
• Another unique restriction enzyme site is the SalI site located in exon 3.
• the human BSSL variant genomic sequence was inserted between these sites, Kpnl and SalI, by the following strategy: First, pS314 was digested with Kpnl and SalI and a fragment representing the cleaved plasmid was electrophoretically isolated. Second, pS312 was digested with Kpnl and BamHI and a approximately 4.7 kb fragment representing the 5'-part of the human BSSL gene was isolated. Third, pS313 was digested with BamHI and SalI and the 3' -part of the human BSSL gene was isolated. These three fragments were ligated, transformed into competent E. coli bacteria and transformants were isolated after ampicillin selection.
• Plasmids were prepared from several transformants and carefully analyzed by restriction enzyme mapping and sequence analysis.
• One plasmid representing the desired expression vector was defined and designated pS317 (Fig.15).
• pS317 was digested with Norl.
• the recombinant vector element consisting of murine WAP sequence flanking the human BSSL variant genomic fragment was then isolated by agarose electrophoresis. The isolated fragment was further purified using electroelution, before it was injected into mouse embryos.
• a Notl fragment was isolated from the plasmid pS317 according to section 3.1. This DNA fragment contained the murine WAP promoter linked to a genomic sequence encoding human BSSL variant. The isolated fragment, at a concentration of 3 ng/ ⁇ l, was injected into the pronudeus of 350
• C57Bl/6JxCBA/2J-f 1 animals were obtained from Bomholtg ⁇ rd Breeding and Research Centre LTD, Ry, Denmark. After collection of the embryos from the oviductsm, they were separated from the cumulus cells by treatment with hyaluronidase in the medium M2 (Hogan et al., 1986). After washing the embryos were transferred to the medium Ml 6 (Hogan et al., 1986) and kept in an incubator with 5% CO 2 -atmosphere. The injections were performed in a microdrop of M2 under light paraffin oil using
• Transgenic mice were identified by analysis of DNA which has been prepared from exdsed tail samples. The tissue samples were incubated with proteinase K and phenol/ chloroform extracted. The isolated DNA was used in polymerase chain reactions with primers which amplify specific fragments if the heterologous introduced DNA representing the expression vector fragment is present. The animals were also analyzed by DNA hybridization experiments to confirm PCR data and to test for possible rearrangements, structure of the integrated vector elements and to obtain information about the copy number of integrated vector elements.
• mice were analyzed with the two methods and the results demonstrated that 1 mice was carrying the heterologous DNA vector element derived from pS317.
• the result from the PCR analysis and the hybridization experiments were identical (Fig. 17). In total, 10 of 65 tested animals were found to be transgenic for pS317.
• the mouse identified to carry vector DNA element (founder animal) was then mated and the F1 litter was analyzed for transgene by the same procedures.
• RNA isolated from various tissues of pS317 transgenic females during lactation have been separated by agarose formaldehyde gel electrophoresis, blotted to membranes and hybridized with 32 P-labelled BSSL cDNA as a probe. The obtained results show that the expression is restricted to the mammary gland during lactation (Fig. 18).
• Milk samples were collected from the anesthetized founder animal treated with oxytodn to induce lactation and analyzed for the presence of recombinant human BSSL variant. This was done by SDS-PAGE, transfer to nitrocellulose membranes and incubation with polyclonal antibodies generated against native human BSSL. The obtained results demonstrated expression of recombinant human BSSL variant in milk from transgenic mice.
• Figure 19 demonstrates presence of recombinant human BSSL variant in milk from transgenic mice.
• Stable lines of transgenic animals are generated.
• transgenic animals such as rabbits, cows or sheep capable of expressing human BSSL variants may be prepared.
• FL denotes the full-length BSSL.
• the active site is indicated by a cirde and the site for the potential N-linked carbohydrate is indicated by a triangle.
• the region containing the repeats is indicated as a striped area and the conserved C-terminal as a filled area.
• DNA prepared from cell lines expressing full-length BSSL (FL), variant A (A), variant B (B), variant C (C) and variant N (N) were analyzed. 5 ⁇ g of the respective prepared cell derived DNA (left) and 1 ng of purified bacteria derived vector DNA (right), were digested with BamHI. The DNA samples were separated on an agarose gel, transferred to GeneScreen Plus membrane and hybridized with 32 P-labelled human BSSL cDNA.
• RNA from isolated cell lines expressing recombinant BSSL variants 10 ⁇ g of total RNA prepared from cell lines produdng full-length BSSL (FL),variant A (A),variant B (B), variant C (C), variant N (N) were analyzed.
• Filters were hybridized with 32 P-labelled BSSL cDNA. The filter was then rehybridized with a murine ⁇ -actin cDNA probe.
• the ⁇ -actin mRNA signals (lower panels) were used as an internal control for the amounts of RNA loaded onto each lane.
• C127 cells were transfected with different BSSL-constructs: full-length BSSL (FL), variant N (N), variant C (C), variant B (B), variant A (A). After the initial growth period individual dones were selected and allowed to grow until confluency. The number of selected dones (n) are indicated in the figure. Lipase activity was determined on the conditioned media. Values are expressed as ⁇ mol free fatty add released x min -1 x ml of conditioned medium -1 .
• the amounts of lipase activity, expressed as ⁇ mol fatty add released x min -1 , applied to the gel was: Full-length 0.2 (lane 1), variant N 0.16 (lane 2), variant C 0.6 (lane 3), variant B 0.8 (lane 4) and native BSSL 0.1 (lane 5).
• the antiserum used was raised in rabbit against BSSL purified from human milk. The position of size markers (Prestained SDS-PAGE
• Bile salt-dependency of full-length and mutated BSSL Lipase activity was determined in the presence of varying concentrations of sodium cholate (solid lines) or sodium deoxycholate (broken lines) on conditioned media from full-length recombinant BSSL (*), variant A ( ⁇ ), variant B ( ⁇ ), variant C ( ⁇ ), variant N (•) and purified human milk BSSL (O).
• a variant conditioned medium was concentrated on Blue Sepharose as described under Experimental procedures. The amount of the respective enzyme source was chosen to obtain the same level of maximal activity except for variant A which had a maximal activity of only one-tenth of the others. Control experiments showed that the growth media did not influence the level of activity or the bile salt dependency of native BSSL (data not shown).
• peripheral preparations and 7-10 represent the same culture volume making the stain proportional to the production level.
• Lane 1 Pharmacia molecular size markers; Lanes 2 and 8, strain JM109(DE3), induced; Lanes 3 and 7, strain JM109(DE3), not induced;
• Lanes 4 and 10 strain BL21(DE3)pLysS, induced; Lanes 5 and 9, strain BL21(DE3)pLysS, not induced; Lane 6, 25 ng of purified native milk BSSL.
• BSSL variants by sodium cholate Purified preparations of recombinant full-length BSSL (•), recombinant BSSL variants B (O), C ( ⁇ ) and N ( ⁇ ), and purified native milk BSSL ( ⁇ ) were assayed for lipase activity with different concentrations of sodium cholate in the absence (left panel) and in the presence of 5 mM (centre panel) or 10 mM (right panel) deoxycholate.
• recombinant full-length BSSL, BSSL variants and native milk BSSL were incubated at the temperatures indicated in 50 mM Tris-Cl buffer, pH 7.5.
• bovine serum albumin (BSA) was added to 1 mg/ml. After 30 min samples were withdrawn and assayed for lipase activity. Activities are expressed as per cent of the activity for each sample at 0 min. For explanation of symbols, see the legend to Fig. 9.
• A Schematic representation of the localization of PCR-primers used for identification of transgenic animals.
• the 5'-primer is positioned within the WAP sequence starting at the position -148 bp upstream of the fusion between the WAP and BSSL variant.
• the 3'-primer is localized in the first BSSL variant intron ending 400 bp downstream of the fusion point.
• RNA sizes in nucleotides are indicated to the left.
• Hamosh M., Freed, L.M., York, CM., Sturman, J.A., and Hamosh, P.
• MOLECULE TYPE DNA (genomic)

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